DOI: https://doi.org/10.52973/rcfcv-e32150
Received: 17/05/2022 Accepted: 18/06/2022 Published: 05/08/2022
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Revista Cientíca, FCV-LUZ / Vol. XXXII, rcfcv-e32150, 1 - 8
ABSTRACT
Colorectal Cancer (CRC) is dened as colon and rectum cancer and is
among the major causes of mortality in developed Countries. Tarantula
cubensis alcoholic extract (TCAE) and Nerium oleander distillate
(NOD) are reported to have anticancer and antioxidative activity. In
this study, it was aimed to research the impact on cell proliferation
markers of TCAE and NOD given simultaneously in experimental
colon cancer. A total of 24 rats, 6 in each group, were used in the
study. Cancer Control (CC): Azoxymethane was administered at the
beginning of the experiment at a dose of 15 miligrams (mg)· kilograms
-1
(kg), (Subcutaneous, SC) twice, with an interval of a week (wk), to
induce cancer. CC+TCAE: the dosage of Azoxymethane administered
was 15 mg·kg
-1
(SC) twice a wk at the beginning of the experiment,
while in the case of TCAE, it was 0.2 mL·kg
-1
(SC) once a wk for 18
wk from the beginning of the experiment. Fifteen mg·kg
-1
(SC) of
Azoxymethane was administered twice at one-wk intervals at the
beginning of the experiment to the CC+NOD group, and NO distillate
(NOD) was given with water throughout the experiment. Afterwards,
animals were euthanized under appropriate conditions, paran
blocks formed from colon tissues, histochemical AgNOR (Silver-
stained nucleolar organizer regions), and immunohistochemical PCNA
(proliferating cell nuclear antigen) stainings were performed. In the
study, immunohistochemically, PCNA scores and AgNOR count per
nucleus (AgNCI) were signicantly decreased in C-TCAE and C-NOD
groups (P<0.001). AgNOR Area index (AgNAI) (P<0.01), Core Area Index
(CAI) (P<0.05), and AgNOR Area index/Core Area Index (AgNAI/CAI)
(P<0.01) scores were signicantly decreased in the C-TCAE group. As
a result, it was concluded that both TCAE and NOD are effective as
chemopreventive drugs and that TCAE presents a more pronounced
antiproliferative effect than NOD.
Key words: Azoxymethane; AgNOR; immunohistochemistry; PCNA;
pathology
RESUMEN
El cáncer colorrectal (CCR) se dene como el cáncer de colon y recto,
y se encuentra entre las principales causas de mortalidad en los países
desarrollados. Se informa que el extracto alcohólico de Tarántula
cubensis (TCAE) y el destilado de Nerium oleander (NOD) tienen actividad
anticancerígena y antioxidante. En este estudio, el objetivo fue
investigar los efectos de TCAE y NOD administrados simultáneamente
sobre los marcadores de proliferación celular en el cáncer de colon
experimental. En el estudio se utilizaron un total de 24 ratas, 6 en cada
grupo. Control del Cáncer (CC). se administró azoximetano al comienzo
del experimento a una dosis de 15 miligramos (mg)·kilogramos
-1
(kg)
(subcutánea, SC) dos veces, con una semana (sem) de diferencia, para
inducir el cáncer. CC+TCAE: la dosis de Azoximetano administrada
fue de 15 mg·kg
-1
(SC) dos veces por sem al inicio del experimento,
mientras que en el caso de TCAE fue de 0,2 mL·kg
-1
(SC) una vez por
sem durante 18 sem desde el inicio del experimento. Se administraron
15 mg·kg
-1
(SC) de azoximetano dos veces a intervalos de una sem al
comienzo del experimento al grupo CC+NOD, y se administró destilado
de NO (NOD) con agua durante todo el experimento. Posteriormente,
los animales fueron sacrificados en condiciones apropiadas, se
realizaron bloques de parana formados a partir de tejidos de colon,
AgNOR histoquímico (regiones organizadoras nucleolares teñidas
con plata) e inmunohistoquímico PCNA (antígeno nuclear de células
proliferantes) se realizaron. En el estudio, inmunohistoquímicamente,
las puntuaciones de PCNA y el recuento de AgNOR por núcleo (AgNCI) se
redujeron signicativamente en los grupos C-TCAE y C-NOD (P<0,001).
Las puntuaciones del índice de área AgNOR (AgNAI) (P<0,01), el índice
de área central (CAI) (P<0,05) y el índice de área AgNOR/índice de área
central (AgNAI/CAI) (P<0,01) se redujeron signicativamente en el
C-TCAE grupo. Como resultado se concluyó que, tanto TCAE como NOD
son efectivos como fármacos quimiopreventivos y que TCAE presenta
un efecto antiproliferativo más pronunciado que NOD.
Palabras clave: Azoximetano; AgNOR; inmunohistoquímica; PCNA;
patología
Effect of Tarantula cubensis alcoholic extract and Nerium oleander distillate
on cell proliferation markers in colon carcinogenesis
Efecto del extracto alcohólico de Tarantula cubensis y el destilado de Nerium oleander sobre los
marcadores de proliferación celular en la carcinogénesis de colon
Ozgur Ozdemir
1
, Gokhan Akcakavak
2
* and Mehmet Tuzcu
1
1
Selcuk University, Faculty of Veterinary Medicine, Department of Pathology. Selcuklu, Konya, Turkey.
2
Yozgat Bozok University, Faculty of Veterinary Medicine, Department of Pathology, Sorgun, Yozgat, Turkey.
*Email: gokhan.akcakavak@bozok.edu.tr
Tarantula cubensis extract and Nerium oleander distillate in colon carcinogenesis markers / Ozdemir et al. _______________________________
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INTRODUCTION
Colorectal cancer (CRC) is dened as colon and rectum cancer [32].
Colon Cancer (CC), also known as CRC or bowel cancer, is used to
describe neoplasia in the colon, rectum or cecum [43]. The incidence
of CRC as one of the principal causes of death related to cancer
around the globe, it has been increasing in recent years [27, 45].
Nowadays, one million people in the world are diagnosed with CRC,
and it is foreseen that it causes the death of approximately half of
them. In addition, among all the types of cancer, the death toll caused
by CRC rises to 10% [16].
Tarantula cubensis alcoholic extract (TCAE) is frequently used in
the Veterinary eld as a homoeopathic product. TCAE is reported to,
thanks to its effect on epithelialization, accelerate wound healing, show
anti-inammatory effects, induce the demarcation of necrotic tissues
and have anti-oedematous effects [21, 24]. It has been reported that
its use on tumours such as oral papillomatosis in dogs (Canis familiaris)
and mammary tumours had successful results [21, 26].
Nerium oleander (NO, oleander) is cultivated as an fancy plant in
parks and gardens in temperate and subtropical regions worldwide.
Since all parts of the NO plant are poisonous and cause cardiotoxicity,
its use for medical purposes is limited [8]. Preceding studies have
pointed out that NO possesses anticancer and antidiabetic effects
[7, 42]. Although NO extracts contain some toxic elements, different
reports highlight the absence of toxicity in rats (Rattus norvegicus) in
acute oral toxicity studies where NO distillate (NOD) was administered
orally, which is in line with the recommendations of the Organisation
for Economic Co-operation and Development (OECD). In the study
performed by Bas et al. [7], it was expressed that after 2 weeks (wk),
it did not show undesirable effects on biochemical parameters,
macroscopically and microscopically. In addition, it was reported
that Nerium oleander distillate (NOD) did not have any toxic effects
after acute and subchronic administration to rats [14, 28].
Proliferating cell nuclear antigen (PCNA) is the term used to refer
to a nuclear protein that plays a role in the regulation of the cell
cycle as well as in the synthesis of deoxyribonucleic acid (DNA),
and its expression takes place in the nuclei of cells in proliferation,
especially in the course of the phases G1 and S [46]. PCNA begins
to be synthesized in phase G1, reaching the highest point in phase
S and decreasing considerably in the M and G2 phases [31]. In this
context, PCNA, because of its reection of the proliferation activity
in the cell, is widely used in healthy and tumoral tissues as a marker
of cell proliferation [38]. In addition, as PCNA is closely connected
to the biological activity of tumour cells, it is foreseen that it plays
an effective role in tumorigenesis. There is a optimistic correlation
among PCNA expression and malignancy [13, 44].
Nucleolus organizer regions (NOR) are chromosomal loops of
DNA which play a role in ribosomal synthesis [20]. The acrocentric
chromosomes 13, 14, 15, 21, and 22 are, in their brief arms, the location
place of the NORs. The argyrophilic proteins AgNORs, on the other
hand, can be detected via silver nitrate staining the NORs and can be
easily described as dark brown or black spots, especially in the nuclear
area [5]. It is suggested that the cell activation may be deduced from
the count of AgNOR points in a nucleus; thus, the activity may be
assessed by this index. Being an increased AgNOR count an indicator
of increased cellular activity compared to protein synthesis, it is
reported to be associated with neoplastic changes [37]. Although
an average of 20 black AgNOR spots are seen in a normal cell, the
number of AgNORs increases in parallel with the increasing amount
of DNA in dysplastic or malignant cells [5]. It has been reported that
AgNOR measurements in animal skin tumours increase in malignant
tumours, and AgNOR number and area indices can be considered
important markers in determining malignancy [25].
PCNA and AgNOR proliferation indices are frequently used to evaluate
the chemopreventive effect of certain substances in experimental
models of colon cancer [3, 4]. The aim of this study aimed to
determine the effects of simultaneous administration of TCAE and
NOD as chemopreventive on PCNA and AgNOR proliferative indices
in Azoxymethane (AOM) induced colon cancer in rats.
MATERIAL AND METHODS
Animal material
The material of the study was constituted of 24 rat intestinal (colon)
paran blocks (TABLE I), which were obtained from the study named
“Ecacy of Tarantula cubensis alcoholic extract and Nerium oleander
distillate in experimental colon cancer” Er et al. [15]. The study was
approved by SUVDAMEK (Decision number: 2021/113).
Study design
In the study, 4 groups of rats were created. While 8 rats formed the
control group, the other 30 rats were divided into 3 groups equally.
Cancer Control (CC): in order to induce cancer, Azoxymethane was
administered at the beginning of the experiment at a dose of 15 mg·kg
-1
subcutaneous (SC) twice, with an interval of a wk. CC+TCAE: the dosage
of AOM administered was 15 mg·kg
-1
(SC) twice a wk at the beginning
of the experiment, while in the case of TCAE, it was 0.2 mL·kg
-1
(SC)
once a wk for 18 wk from the beginning of the experiment. Fifteen
mg·kg
-1
(SC) of AOM was administered twice at one-wk intervals at the
beginning of the experiment to the CC+NOD group, and NOD was given
with water throughout the study. At the end of the 18th wk, anaesthesia
was implemented to the rats in a dose of 95 mg·kg
-1
(SC) ketamine + 5
mg·kg
-1
(SC), xylazine and euthanasia was performed by the cervical
dislocation method [15]. A method described in the literature was
used to obtain NOD [7]. In this study, the paran blocks of intestinal
samples (colon tissue of 24 rats in total, 6 in each group) taken from the
study, the design of which was described above, were used (TABLE I).
TABLE I
Number of rats used in the study
Groups C CC C-TCAE C-NOD
Number of rats (n) 6 6 6 6
C: Control, CC; Cancer Control, C-TCAE: Cancer+TCAE, C-NOD: Cancer+NOD
PCNA Immunohistochemistry staining
After 4-5 milimicras (µm) sections from those paran blocks that
best represented the tumour were taken to adhesive slides, they were
kept in an incubator (Binder ED 56, Darmstadt, Germany) at 60°C for
20 minutes (min). Afterwards, paran extraction and rehydration
processes were performed on the sections. Immunohistochemistry
(IHC) staining was performed with the UltraVision Detection System
Anti-Polyvalent, HRP (Horseradish Peroksidaz) (Ready-To-Use,
TP-060-HL, Lab Vision, USA) IHC kit following the manufacturers
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recommendations. The antigen was recovered by treating the tissues
in a microwave oven (UTD-1420, Utest, Turkey) with Citrate buffer
(pH6) solution for 20 min at 750 watts. Mouse monoclonal Anti-PCNA
antibodies (Dako, clone PC10, Cat No; M0879, 1-hour (h) incubation
were used as primer and 3,3 diaminobenzidine (DAB) as chromogen.
The incubation of sections with phosphate buffered saline (PBS) rather
than primary antibodies was used to obtain the negative controls. After
counterstaining with Mayers Hematoxylin, it was passed through alcohol
and xylene series, covered with a coverslip and examined under a light
microscope (Olympus BX51, Tokyo, Japan). The Allred scoring method
was used for the extent and intensity of staining in the IHC scoring of the
sections [23]. Staining intensity score (0; absent, 1; weak, 2; moderate,
3; strong) and staining extent (0: absent; 1: >0-1/100; 2: > 1/100-1/10; 3:;
>1/10-1/3; 4:1/3-2/3 and 5: >2/3-1) total scores were obtained.
AgNOR staining and quantication
From the paran blocks that best represent the tumour, slides
were cut with 4-5 μm thickness and stained for AgNOR, according
to Hatipoglu et al. [25]. Tissues were passed, in order, through xylol,
alcohol and distilled water series. Afterwards, mixing 1 unit of 2% gelatin
with 1% aqueous formic acid solution and 2 units of 50% aqueous AgNO3
solution resulted in a mixture which was ltered through a 0.2 µm lter
in the dark. Ensuing, the tissues were incubated in the existing mixture
at 37°C for 25 min. Then bidistilled water was used to treat the tissues,
and they were kept for 5 min in 5% sodium thiosulfate. Afterwards, the
tissues were passed through alcohol, xylene and bidistilled water and
adhered with synthetic adhesive. The examination of the dysplastic
crypts was performed under a light microscope using an immersion
objective (1000X magnication). Photos that would later be transferred
to a computer were taken from the areas with dysplastic crypts with a
digital imaging system (Olympus, DP12-BSW, version 01.03, Olympus,
Tokyo, Japan). The evaluation was performed on 25 dysplastic crypt
epithelium using the image analysis program (Digital Life Science
Imaging, analySIS® LS Starter, 2.2, Build 1110, An Olympus Company,
Munster, Germany). The following indices were calculated from each
case; AgNOR Area Index (AgNAI) (Total NOR area/25), Core Area Index
(CAI) (Total Core Area/25), AgNOR Count Index (AgNCI) (Total NOR Point
Count/25), AgNAI/CAI index (AgNOR Area Index/Core Area Index).
Statistical analysis
IHC and AgNOR data obtained in the study were evaluated with
One-way ANOVA and Duncan test as Post hoc test (SPSS,Inc., Chicago,
IL, USA 25.0). A value of P<0.05 was considered statistically signicant.
TABLE II
Distribution of AgNOR indices and PCNA indices into groups (Mean ± SD)
C (n = 6) CC (n = 6) C-TCAE (n = 6) C-NOD (n = 6) P-value
AgNAI 1.82 ± 0.23
b
3.89 ± 0.64
a
2.78 ± 0.82
b
3.92 ± 1.39
a
<0.01
CAI 18.23 ± 3.34
c
23.57 ± 3.14
ab
20.96 ± 3.46
bc
26.06 ± 4.67
a
<0.05
AgNCI 1.82 ± 0.28
c
3.44 ± 0.33
a
2.03 ± 0.18
c
2.42 ± 0.19
b
<0.001
AgNAI/CAI 0.10 ± 0.02
c
0.17 ± 0,03
a
0.13 ± 0.02
bc
0.15 ± 0.03
ab
<0.01
PCNA 3,83 ± 1.17
c
7,50 ± 0,84
a
5,83 ± 0,41
b
5,83 ± 0,75
b
<0.001
a,b,c
: Indicates statistical signicance between groups (C, CC, C-TCAE, C-NOD). (C: Control, CC: Cancer Control, C-TCAE:
Cancer+TCAE, C-NOD: Cancer+NOD, AgNAI: AgNOR Area Index, CAI: Core Area Index: AgNCI; AgNOR Count Index,
AgNAI/CAI: AgNOR Area Index/Core Area Index, PCNA: Proliferating cell nuclear antigen, AgNOR: Silver-stained
nucleolar organizer regions)
RESULTS AND DISCUSSION
Statistical scores of PCNA and AgNOR analysis in control and
experimental animals are shown in TABLE II. Regarding PCNA staining,
the highest scores were found in the experimental groups. Especially
in the experimental CC induced by AOM, the PCNA expression was
increased by the intense nuclear staining (FIGS. 1-2, P<0.001). PCNA
stainings showed an intense expression mostly in dysplastic crypts
(FIG. 2). A signicant decrease in PCNA nuclear expression was
detected in the C-TCAE and C-NOD groups in comparison with the
CC group (FIGS. 1-2, P<0.001).
Regarding AgNOR staining, black-brown NORs were observed in all
control and experimental groups (FIG. 3). It was determined that all
the indices in the experimental groups were increased in comparison
with the Control group (C). When the AgNOR Area Index (AgNAI) was
evaluated, the highest scores were found similar in the C-NOD group
and the CC group. In the C-TCAE group, the scores decreased compared
to the CC group and were similar to the Control group (C) (P<0.01). In the
Core Area Index (CAI), the highest score was again in the C-NOD group,
being statistically similar to the CC group, and the C-TCAE group had a
lower score (P<0.05) than the C-NOD group, although C and CC group
were similar. In the evaluation of the AgNOR Count Index (AgNCI), the
scores in the CC group were found to be signicantly higher (P<0.001)
in comparison with the other groups. The scores of the C-TCAE and
C-NOD groups decreased, and the C-TCAE group was found closer to
the C group. Although the effect of AgNAI/CAI chemopreventives was
statistically similar, it was observed that the C-TCAE group was similar
to the C group and was statistically signicant (P<0.01).
CRC is, considering its mortality and morbidity, a very important
disease in developed Countries. Although classical treatment methods
are used in the treatment of CRC today, different treatment options
are sought because of the many side effects in individuals [2, 45].
Experimental CRC models induced with chemicals are clinically and
pathologically similar to human CRC [35]. In this context, AOM-induced
experimental colonic carcinogenesis is known as a reliable model that
is frequently used today to evaluate the chemopreventive effect of
many drugs [4, 17]. In the present study, the effects of simultaneous
TCAE and NOD administration on PCNA and AgNOR proliferative
indices were determined in AOM-induced colon cancer in rats.
PCNA is a protein whose IHC determination is frequently used to
examine the proliferation activity in the process of carcinogenesis.
It is especially used as an intermediate marker in chemoprevention
studies of colon cancer carcinogenesis, and its expression levels




Nerium oleander distillate)
Tarantula cubensis extract and Nerium oleander distillate in colon carcinogenesis markers / Ozdemir et al. _______________________________
4 of 8
are examined in many studies [3, 4]. In this study, it was determined
that, in according to the control group, the PCNA expression in the
experimental groups was increased. In the C-TCAE and C-NOD groups,
the scores were found to be signicantly reduced compared to the
CC group (TABLE II, P<0.001). In the study, the severe increase in
PCNA immunoreactivity in hyperplastic and dysplastic crypts in
experimental groups, especially in the CC group, can be interpreted
as a correlation between proliferative activity and hyperplasia and
dysplasia. The ndings of this study show parallelism with the ndings
of chemical-induced experimental colon cancer or colorectal cancer
studies in the literature [3, 4, 33].
Growth factors can regulate the stabilization of gene and protein
expressions of PCNA. Changes in the concentrations of growth
factors and receptor levels, or their combinations, can affect PCNA
expressions in some hyperplastic polyps [10]. Especially Nuclear EGFR
(Epidermal Growth Factor Receptor) can induce or stabilize DNA-PK
(DNA-dependent protein kinase) and PCNA to increase DNA repair and
replication [9]. In addition, it has been reported that the induction of
PCNA tyrosine phosphorylation by EGFR inhibits MMR (DNA mismatch
repair) so that mismatches occur during DNA synthesis [29]. In a study
by Fichera et al. [17], it was announced that EGFR signals increased in
an experimental study induced by AOM. In the present study, the high
scores of PCNA expression in the cancer model induced by AOM, the
CC group, can be interpreted as a result of increased DNA replication
or the formation of DNA mutations. In addition, the decrease of the
PCNA scores in C-TCAE and C-NOD groups leads to the possibility of
a positive effect on EGFR signalling.
In the study performed by Ghasemi-Dizgah et al. [19], it was
announced that TCAE reduces free oxygen radicals and can activate
apoptosis by inducing the caspase-3 pathway in vitro. In addition, in a
study conducted on different cancer cell lines in vitro, it was reported
that TCAE is an apoptosis-inducing agent [39]. In a different study,
it was reported that after treatment with TCAE in canine mammary
tumours, Bcl-2 (B cell lymphoma-2) and Ki-67 expressions were IHC
reduced [21]. Guzińska-Ustymowicz et al. [22] reported that there
is a positive correlation between PCNA and Bcl-2 in CRC in humans.
Also, studies have reported a negative correlation between PCNA
expressions and p53 [6, 41]. In particular, Umesalma et al. [41] attributed
decreased PCNA expression to increased p53 expression in their
study. In addition, in a different study induced by AOM/DSS, it was
reported that messenger Ribonucleic acid (mRNA) expression levels
of p53 and Bax decreased while Bcl-2 expression increased [34]. In

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
Nerium oleander distillate)
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this context, although in this study, no ndings of the expressions of
Bcl-2 and p53 proteins were detected, in the light of the literature,
it can be interpreted that the cells are directed to apoptosis as a
result of decreased PCNA expressions by means of increased p53
and decreased Bcl-2 expressions. In the present study and in the
light of the literature, it is possible to think that the increase in p53
expression and the decrease in Bcl-2 expression caused by TCAE may
be behind the PCNA scores decrease in the C-TCAE group.
Known as a tumor suppressor gene, p21 has functions as a cell
cycle inhibitor and antiproliferative effector in normal cells. These
functions are based on its function as cyclin-dependent kinase (CDK)-
cyclin complexes and PCNA inhibitor as a mediator of p53 tumour
suppressor activity [1]. In one study, it was reported that spider
Macrothele raveni venom increased p21 mRNA and protein expressions
in human hepatocellular carcinoma in vitro HepG2 cell line, and HepG2
cells were directed to apoptosis. They also attributed this to the G2/M
phase cell cycle arrest [18]. Due to the phylogenetic susceptibility of
Tarantula cubensis and Macrothele revani, the cytotoxic functions and
mechanisms of their venoms may be similar. In another study, an in
vitro CRC study with Odoroside, an active ingredient obtained from NO
leaves, reported that Odoroside A treatment stopped the cell cycle in
CRC in the G2/M phase due to increased p21 expression levels. It has
also been reported that it is directed to apoptosis in a p53-dependent
manner, which is associated with the activation of the p21 protein [11].
In a different in vitro CRC study, it has been reported that NO inhibits
cell proliferation by stopping cells in the G2/M phase. It is also stated
to induce apoptosis in cancer cells [30]. In this context, the decrease
in PCNA expression levels in K-TCAE and K-NOD groups may also be
caused by its activity on p21. More molecular studies are needed in
the future to reveal the reason for the decreased PCNA expressions
in the K-TCAE and K-NOD groups in this study.
AgNOR proteins present various morphological changes in the
number and size that are eligible for being used as a marker of cell
proliferation. In this context, it is considered a predictive prognostic
marker for cancer proliferation [33]. In addition, AgNOR image analysis
is a useful method in the evaluation of the cell proliferation rate [5].
Previous studies have announced that the increase in the number of
AgNORs is signicantly higher in malignant cells than in normal cells,
and it is interpreted as related to ribosomal RNA transcription rate, cell
proliferation and DNA ploidy. Additionally, it has been reported that